Abstract

Pluripotent embryonic stem cells (ESCs) are a potential source for cell-based tissue engineering and regenerative medicine applications, but their translation into clinical use will require efficient and robust methods for promoting differentiation. Fluid shear stress, which can be readily incorporated into scalable bioreactors, may be one solution for promoting endothelial and hematopoietic phenotypes from ESCs. Here we applied laminar shear stress to differentiating ESCs using a 2D adherent parallel plate configuration to systematically investigate the effects of several mechanical parameters. Treatment similarly promoted endothelial and hematopoietic differentiation for shear stress magnitudes ranging from 1.5 to 15 dyne/cm(2) and for cells seeded on collagen-, fibronectin- or laminin-coated surfaces. Extension of the treatment duration consistently induced an endothelial response, but application at later stages of differentiation was less effective at promoting hematopoietic phenotypes. Furthermore, inhibition of the FLK1 protein (a VEGF receptor) neutralized the effects of shear stress, implicating the membrane protein as a critical mediator of both endothelial and hematopoietic differentiation by applied shear. Using a systematic approach, studies such as these help elucidate the mechanisms involved in force-mediated stem cell differentiation and inform scalable bioprocesses for cellular therapies.

Cells were seeded on slides coated with collagen type-IV, cultured under static conditions for two days, and then for two days either exposed to 1.5, 5.0, or 15 dyne/cm2 of shear stress (SHEAR samples) or maintained in culture under static conditions (STATIC controls). flk1, tie2, and runx1 gene expression was assessed for SHEAR samples (■) and normalized to trial-matched STATIC controls (□). Data presented are mean ± SEM (n=7-12), where significant differences between STATIC and SHEAR groups are indicated with asterisks (* p<0.05, ** p<0.01, and *** p<0.001).

Cells were seeded on slides coated with collagen type-IV (COL), fibronectin (FN), or laminin (LM), cultured under static conditions for two days, and then for two days either exposed to 5.0 dyne/cm2 of shear stress (SHEAR samples) or maintained in culture under static conditions (STATIC controls). Phase images of cells for both STATIC controls and SHEAR samples on COL, FN, and LM are shown. flk1, tie2, and runx1 gene expression was assessed for SHEAR samples (■) and normalized to trial-matched STATIC controls (□). Data presented are mean ± SEM (n=7-12), where significant differences between STATIC and SHEAR groups are indicated with asterisks (** p<0.01 and *** p<0.001). Scale bar represents 100Lm.

Samples were exposed to four days of either STATIC (white striped bars) or SHEAR treatment (τ=5.0 dyne/cm2: black striped bars). A representative histogram of FLK1 protein expression (A) is shown for an immunostaining 2° antibody-only control (filled gray), a STATIC experimental control (gray line), and a SHEAR experimental sample (black line). Fluorescence of both STATIC and SHEAR samples were assessed and compared to flow cytometry control samples to calculate the percentage of cells positive for markers of early differentiation. Assessed proteins were FLK1 (B), TIE2 (C), and RUNX1 (D). Data presented are mean ± SEM (n=3-4), where significant differences between STATIC and SHEAR groups are indicated by asterisks (* p<0.05, *** p<0.001)

Samples were exposed to four days of either STATIC (white bars, striped or dotted) or SHEAR treatment (τ=5.0 dyne/cm2: black bars, striped or dotted). Fluorescence of both STATIC and SHEAR samples were assessed and compared to flow cytometry control samples to calculate the percentage of cells positive for differentiation markers. Assessed proteins were PECAM1 (A) and CD41 (B, LEFT). A representative histogram of NOS3 and VWF protein expression (C) is shown for an immunostaining 2° antibody-only control (filled gray), a STATIC experimental control (gray line), and a SHEAR experimental sample (black line). To detect subtle changes in protein expression, quartile analysis was used to quantify the expression of NOS3 and VWF. Following STATIC and SHEAR treatment, hematopoietic potential was assessed using a colony counting assay (B, RIGHT). Data presented are mean ± SEM (n=3-4), where significant differences between STATIC and SHEAR groups are indicated by asterisks (* p<0.05) or displayed p-value.

Cells were cultured under static conditions for 1-3 days before one day of STATIC (□) or SHEAR (τ=15 dyne/cm2, ■) treatment. Gene expression (normalized to gapdh) was calculated for flk1 (A), tie2 (B), and runx1 (C). Data presented are mean ± SEM (n=6-8), where significant differences between trial-matched STATIC and SHEAR groups are indicated by asterisks (*=p<0.05, **=p<0.01).

Delayed exposure to shear stress is less effective at promoting a hematopoietic phenotype

Cells were cultured under static conditions for 2 or 3 days before four days of either STATIC (striped white bars) or SHEAR (τ=5.0 dyne/cm2, striped black bars) treatment. Fluorescence of both STATIC and SHEAR samples were assessed and compared to flow cytometry control samples to calculate the percentage of cells positive for markers of differentiation. Assessed proteins were FLK1 (A), TIE2 (B), RUNX1 (C), and CD41 (D). Data presented are mean ± SEM (n=3-4), where significant differences between STATIC and SHEAR groups were determined by a t-test and indicated by asterisks (* p<0.05 and *** p<0.001).

FLK1 inhibition negates the effects of shear stress on early differentiation

Cells were exposed to two days of STATIC (□) or SHEAR (τ=5.0 dyne/cm2, ■) treatment, where media from select STATIC and SHEAR samples was supplemented with a FLK1 inhibitor (SU14-8: 4.5Lg/ml) during treatment. Gene expression was assessed and each value was normalized to the average value of the trial-matched STATIC controls. Genes assessed include tie2 (A) and runx1 (B). Data presented are mean ± SEM (n=3-4), where significant differences between trial-matched STATIC and SHEAR are indicated by asterisks (*** p<0.001).